(81f) An Integrated Synthetic Biology Pipeline for Novel Plant Natural Product Discovery in Yeast

Natural products from plants and their derivatives are an abundant source of valuable compounds such as drugs. However, the discovery of plant natural products and their biosynthetic pathways remains challenging, which may be partially due to the large size and complexity of plant genomes, and the difficulties in plant gene isolation and pathway validation. Recent research progress in plant genome sequencing and transcriptomics analysis allows better understanding of plant genomics, which offers the potential to expedite the pathway discovery process. However, there are no approaches to efficiently decode the complex plant genomes and to precisely elucidate these pathways in plants. Here we report an innovative pipeline for systematic and accelerated discovery of novel plant natural products and associated biosynthetic pathways. This versatile pipeline combines leading techniques in bioinformatics, synthetic biology, and analytical chemistry, and enables fast-paced, high-throughput discovery of plant natural products through predicted gene cluster reconstruction and metabolite analysis in heterologous hosts such as Saccharomyces cerevisiae. This approach comprises three main steps: 1) new plant metabolic pathway prediction by bioinformatic tools for gene cluster identification and prioritization, which is inspired by recent bioinformatics analysis revealing large numbers of metabolic pathways clustered in plant genomes; 2) cluster reconstruction in yeast using established synthetic biology toolkits and gene assembly pipeline for heterologous production of novel compounds and functional validation of predicted biosynthetic pathways; 3) novel compound identification by optimized mass spectrometry-based untargeted metabolomics analysis. We demonstrated this approach by discovering novel phenylpropanoid derivatives and elucidating their biosynthetic pathways in yeast, which not only sheds light on multiple branched phenylpropanoid biosynthetic pathways, but also generates engineered yeast strains providing a large amount of pathway intermediates, thereby establishing a platform for advancing drug discovery and production. This integrated approach will support the future development of scalable drug manufacturing platforms, ultimately advancing drug discovery, development, and production in yeast.